This NRSA F30 application requests fellowship support for Ms. Alexis Carulli in the field of gastrointestinal physiology, focusing on the study o intestinal epithelial cell homeostasis and responses to damage and repair. The research project investigates the role of the Notch signaling pathway for regulation of intestinal stem cells (ISCs) Our recent studies have shown that Notch has critical functions for both ISC maintenance as well as downstream cell fate decisions. We have shown that chronic Notch inhibition in adult mice results in increased numbers of secretory cell types, decreased cellular proliferation, and a marked decrease in the ISC marker Olfm4. My preliminary data suggests that the Olfm4 response is Notch1(N1)- specific, suggesting that N1 is the predominate Notch receptor for the ISC. Furthermore, I have shown that acute Notch inhibition leads to transient knockdown of Olfm4, increased secretory cells, and a long-lasting increase in cellular proliferation, a stark contrast to the suppression seen with chronic Notch inhibition. It is clear that Notch signaling plays an important role in the ISC, but the specific cellular responses are unknown. To further understand the underlying cellular dynamics with Notch-ISC regulation, this proposal will test the following hypothesis: The Notch signaling pathway regulates the transition from ISC to fated progenitor cells and loss of Notch signaling leads to depletion of the stem cell pool and activation of quiescent stem cells to replenish the pool. To test this hypothesis both genetic and pharmacologic mouse models of Notch inhibition will be employed. Genetic manipulation and marking of ISC cell populations will take advantage of Cre drivers specific for active or quiescent ISCs. Strains of floxed-gene mice will allow deletion of Notch pathway components in specific ISC cell populations. Three specific aims are proposed: (1) Test the hypothesis that Notch1 receptor signaling is required to maintain ISCs and that both N1 and N2 receptor signaling is required for regulation of cell fate to enterocyte lineages. (2) Test the hypothesis that ISC loss by Notch inhibition leads to activation of a quiescent stem cell to repopulate the active cycling stem and progenitor pool. (3) Develop a mathematical model to determine if Notch signaling regulates asymmetric stem cell division vs. symmetric formation of progenitor cells. United, these aims will provide comprehensive training in mammalian physiology, molecular biology, and mathematical modeling, advancing my career as a physician-scientist. PUBLIC HEALTH RELEVANCE: Intestinal stem cells provide the cellular source for intestinal epithelial homeostasis, growth, and repair, as well as provide a potential source for intestinal cancers. The Notch signaling pathway has been shown to regulate intestinal cell fate and proliferation rates. This project aims to elucidate the role of Notch in regulating the intestinal stem cell to better understand normal intestinal function as well as intestinal diseases, such as cancer, that arise as a consequence of stem cell dysfunction.